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Biochemical study of Polynucleotide Phosphorylase from the foodborne pathogen Campylobacter jejuni
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As bactérias devem adaptar-se e responder rapidamente a diferentes condições ambientais. As ribonucleases são as enzimas responsáveis pela maturação e degradação de moléculas de RNA, permitindo uma rápida adaptação dos níveis de RNA a diferentes ambientes. Esta adaptação é crucial para as bactérias patogénicas invadirem e estabelecerem-se dentro do hospedeiro. A PNPase (Polynucleotide phosphorylase) é uma exoribonuclease homotrimérica com actividade 3’-5’ e que para além de degradar RNA, também é capaz de sintetizar caudas heteropoliméricas. Esta enzima tem sido implicada na virulência em muitos patogénicos humanos, nomeadamente em Salmonella, Dichelobacter nodosus, Dickeya dadantii, Yersinia e Campylobacter jejuni. C. jejuni é um importante patogénico humano de origem alimentar e é considerado como a principal causa de gastroenterite bacteriana em humanos em todo o mundo. No entanto, a informação sobre o metabolismo do RNA em C. jejuni é limitada. Sabe-se que a PNPase é essencial para a sobrevivência das células a baixa temperatura, afecta a síntese de proteínas envolvidas na virulência e tem um papel importante na motilidade, adesão das células/capacidade de invasão e colonização das aves. Para compreender como a PNPase está envolvida na virulência é necessário fazer uma análise bioquímica desta ribonuclease e ver como esta é influenciada por factores físicos e químicos. Neste trabalho caracterizámos a actividade bioquímica da PNPase de C. jejuni. Sobre-expressámos e purificámos a PNPase, os mutantes PNPase_ΔS1 e PNPase_ΔS1ΔKH de C. jejuni e testámos a sua actividade e capacidade de ligação utilizando substratos de RNA sintéticos. Demonstrámos que a actividade da PNPase é regulada em função da temperatura. Além disso, verificámos que as actividades degradativas e de polimerização são altamente reguladas na presença de certos metabolitos. Mostrámos também que os domínios KH e S1 da PNPase desempenham um papel importante na ligação do substrato e na formação de trímeros, com consequências para a actividade da proteína.
Bacteria must adapt and rapidly respond to different environmental conditions. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, enabling a fast adaptation of RNA levels to different environments. This adaptation is crucial to bacterial pathogens invade and establish inside the host. Polynucleotide phosphorylase (PNPase) is a homotrimeric 3’-5’ exoribonuclease that has both degradative and synthetic capabilities. It has been implicated in virulence in many human pathogens, namely in Salmonella, Dichelobacter nodosus, Dickeya dadantii, Yersinia and Campylobacter jejuni. C. jejuni is an important human foodborne pathogen, and is considered as the leading cause of human bacterial gastroenteritis worldwide. However, the information regarding RNA metabolism in this pathogen is limited. It is known that PNPase is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization. A better understanding about C. jejuni PNPase biochemistry and how it is influenced by physical and chemical factors is an approach to understand how this ribonuclease is involved in virulence. In this work we have characterized the biochemical activity of PNPase from C. jejuni. We have overexpressed and purified C. jejuni PNPase and PNPase_ΔS1 and PNPase_ΔS1ΔKH mutants, and tested their activity and binding ability using synthetic RNA substrates. We have demonstrated that PNPase activity is regulated according to the temperature. Moreover, both degradative and polymeric activities are highly regulated in the presence of certain metabolites. We have also shown that both the KH and S1 domains of PNPase play critical roles in substrate binding and trimerization, with consequences for the activity of the protein.
Bacteria must adapt and rapidly respond to different environmental conditions. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, enabling a fast adaptation of RNA levels to different environments. This adaptation is crucial to bacterial pathogens invade and establish inside the host. Polynucleotide phosphorylase (PNPase) is a homotrimeric 3’-5’ exoribonuclease that has both degradative and synthetic capabilities. It has been implicated in virulence in many human pathogens, namely in Salmonella, Dichelobacter nodosus, Dickeya dadantii, Yersinia and Campylobacter jejuni. C. jejuni is an important human foodborne pathogen, and is considered as the leading cause of human bacterial gastroenteritis worldwide. However, the information regarding RNA metabolism in this pathogen is limited. It is known that PNPase is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization. A better understanding about C. jejuni PNPase biochemistry and how it is influenced by physical and chemical factors is an approach to understand how this ribonuclease is involved in virulence. In this work we have characterized the biochemical activity of PNPase from C. jejuni. We have overexpressed and purified C. jejuni PNPase and PNPase_ΔS1 and PNPase_ΔS1ΔKH mutants, and tested their activity and binding ability using synthetic RNA substrates. We have demonstrated that PNPase activity is regulated according to the temperature. Moreover, both degradative and polymeric activities are highly regulated in the presence of certain metabolites. We have also shown that both the KH and S1 domains of PNPase play critical roles in substrate binding and trimerization, with consequences for the activity of the protein.
Descrição
Palavras-chave
Microbiologia médica Bacteriologia Enzima Células Ribonucleases Moleculas de RNA Hospedeiro Polynucleotide phosphorylase
Contexto Educativo
Citação
Editora
Instituto de Higiene e Medicina Tropical